Dysfunction or defects of craniofacial tissues has a significant impact on the patient's life. Therefore, reconstruction of damaged tissues is highly desired. Dental follicle stem cells are a potential source for tissue replacement due to their ability to differentiate into specialized cells, including bone and fat (1). However, fundamental processes in determining the differentiation pathways remain undetermined. Thus, our long-term goal is to characterize the molecular mechanisms controlling stem cell differentiation so that more rigorous and reliable protocols can be established for reconstruction of craniofacial components. Calcium (Ca2+) oscillations are a phenomenon commonly observed during stem cell differentiation and there is evidence that it may be important for directing and terminating the process (2-4). The Transient Receptor Potential Melastatin 4 (TRPM4) channel is essential for Ca2+ oscillations because it controls the amount of Ca2+ entering cells (5). Molecular suppression of TRPM4 increases Ca2+ entry and cytokine production in immune cells (5). In fact, enhanced Ca2+ entry also facilitates the differentiation of stem cells into bone and fat (2, 3). We have identified TRPM4 in stem cells using a molecular approach and demonstrated its functionality with the patch-clamp technique (preliminary data). Our central hypothesis is that TRPM4 represents a key regulatory mechanism that controls Ca2+ entry in stem cells and is a potential target to enhance tissue regeneration. The proposed experiments will determine the role of TRPM4 in dental follicle stem cells. In Aim #1, we will determine whether TRPM4 mediates stem cell differentiation into bone and fat. Stable TRPM4 knockdown clones will be cultured in osteoblast or adipocyte differentiation medium and examined for alkaline phosphatase enzyme activity and for the presence of Ca2+ deposition in the extracellular matrix using Alizarin Red S (osteoblast differentiation) or lipid formation inside intracellular vesicles using Oil Red O (adipocyte differentiation). We will also determine if osteogenic or adipogenic genes are more rapidly turned on/off if TRPM4 is inhibited using RT-PCR and microarray analysis. In Aim #2, we will determine whether TRPM4 controls Ca2+ signals. First, we will perform a detailed quantitative analysis and characterization of TRPM4 using the patch-clamp technique. Second, we will test the hypothesis that inhibition of TRPM4 will enhance Ca2+ entry in stem cells. We will knockdown TRPM4 with two approaches: 1) Use of the shRNA (stable system), and 2) Use of Dominant Negative constructs (transient system). We will assess the impact of TRPM4 knockdown on Ca2+ signals generated by G-protein coupled receptor agonists and spontaneous oscillations using real-time digital imaging analysis. PUBLIC HEALTH RELEVANCE: Stem cell therapy offers a promising approach to providing an advanced and reliable therapeutic strategy to repair craniofacial defects. However, fundamental processes determining stem cell differentiation are not well understood. The overall goal of this research is to understand how calcium signals impact the fate of stem cells (e.g. differentiation into bone, fat, muscle and others). It is well established that ion channels control the shape and frequency of calcium signals. Depending on the calcium pattern, different genes can be turned on or off. In fact, physical manipulation of calcium signals facilitates stem cell differentiation into bone. Calcium signals can also activate transcription factors to make stem cells become fat tissue. Therefore, if we can establish a link between calcium patterns and stem cell fate, it would be possible to generate specialized tissues by targeting ion channels. This may offer an alternative approach to explore the unique properties of stem cells for tissues engineering or patient transplant. The principal investigator has published 17 manuscripts in peer-reviewed scientific journals, including 4 papers on the regulation of calcium signals by TRPM4 in immune and pancreatic [unreadable]-cells and is currently expanding the knowledge obtained to stem cell biology.